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Soluble CD163 (sCD163) is a scavenger receptor shed in serum during inflammatory activation of macrophages. We investigated if sCD163 was increased and predicted outcome in acute liver failure (ALF).
Samples from 100 consecutive patients enrolled in the U.S. ALF Study Group for whom sera were available were collected on days 1 and 3, and clinical data were obtained prospectively. sCD163 levels were determined by ELISA.
The median level of sCD163 was significantly increased in ALF (21.1 mg/l (range 3.6 – 74.9)) as compared to healthy controls (2.3 mg/l (0.65 – 5.6), p < 0.0001) and patients with stable liver cirrhosis (9.8 mg/l (3.6 – 16.9), p=0.0002). sCD163 on day 1 correlated significantly with ALT, AST, bilirubin, and creatinine. sCD163 concentrations on day 3 were elevated in patients with fatal outcome of disease compared to spontaneous survivors, 29.0 mg/l ((7.2 – 54.0) vs. 14.6 mg/l (3.5 – 67.2), respectively (p = 0.0025). Patients that were transplanted had intermediate levels. Sensitivity and specificity at a cut off level of 26 mg/l was 62 % and 81 % respectively.
Activated macrophages are involved in ALF resulting in a 10-fold increase in sCD163. A high level (> 26 mg/l) of sCD163 was significantly correlated with fatal outcome and might be used with other parameters to determine prognosis.
Macrophages and other cells of the immune system are involved in the pathogenesis of acute liver failure (ALF), as reflected by activation of both pro- and anti-inflammatory cascades of the innate immune system (1–4). Recent findings suggest that changes in the inflammatory balance may be important for the disease course, and that a shift towards an immunosuppressive, anti-inflammatory state may be deleterious (1,3).
CD163 is a macrophage lineage-specific scavenger receptor regulated by IL10 and known to be involved in several anti-inflammatory functions of the immune system (5). The receptor is significantly up-regulated on liver CD68+ macrophages during acute hepatitis (6). Highly elevated levels of a circulating form of the receptor (soluble CD163, sCD163) have recently been described in sera from patients with ALF (7), indicating that CD163 positive macrophages could be involved in the pathogenesis and clinical course of ALF.
The difficult decision whether to perform a liver transplantation in ALF must be made early in the disease course (8), and new marker molecules that could be incorporated into robust prognostic models would have important clinical value. In the present work we investigated whether levels of sCD163 were related to clinical outcome when measured early in patients with ALF.
One hundred patients with ALF were prospectively enrolled in the U.S. ALF Study Group (9). Patients met ALF criteria of coagulopathy (International Normalized Ratio > 1.5) and any degree of hepatic encephalopathy. On admission, 45 patients had hepatic coma grade I or II, and 55 patients had grade III or IV hepatic encephalopathy; maximum hepatic encephalopathy was I or II in 37 patients, whereas 63 patients had maximum grade III or IV hepatic encephalopathy. Consecutive patients for whom sera were available were chosen. After informed consent was obtained from next of kin, detailed prospective clinical and laboratory data were entered in anonymous fashion into case report forms at admission to study and 3 weeks later, or at time of death or liver transplantation. The aetiologies were established by the site investigators using standard criteria. Blood samples for sCD163 analyses were taken at the day of enrolment (day 1) and two days later (day 3). Seventy-five samples were obtained at day 1, and 77 samples at day 3. Samples from both days were available from 52 patients. Clinical and biochemical data are summarised in table 1. The study met all requirements of institutional review boards at the respective study sites. One-hundred age and sex-matched controls without known hepatic or biliary disease were selected from 240 samples obtained from the NOBIDA biobase (http://www.furst.no/norip/nobida/index.htm). Seven patients with chronic alcoholism (without clinical symptoms of liver cirrhosis) and 13 patients with stable liver-cirrhosis due to chronic alcoholism (without ascites or esophageal varices) were also included as pathological controls.
sCD163 was measured in ELISA as described in detail (10). In brief, rabbit anti-CD163, 4 mg/l was coated onto micro-titer wells. After wash, 100 μl of sample (diluted 1:50 in PBS with albumin, pH 7.2) was added and incubated for 1 h. The wells were washed and 100 μl of monoclonal anti-CD163 (GHI/61, diluted 1:500) was added and incubated for 1 h. After wash, 100 μl of peroxidase-labeled antibody (goat anti-mouse immunoglobulins, DAKO P447, diluted 1:4000) was added and incubated for 1 h. The wells were washed, and 100 μl of a H2O2/1,2-phenylenediamine dihydrochloride substrate solution was added. After 15 min 50 μL of 1 mol/l H2SO4 was added, and the plates were read at 492/620 nm. Control-samples and standards of purified CD163 were co-analysed in each run. The inter-assay imprecision was 6.2 and 9.7 %CV at levels of 1.8 and 3.9 mg/l respectively (n=15×2).
Non parametric tests were used for comparison of groups (Mann-Whitney test for unpaired data and Wilcoxon signed rank test for paired data) and for correlation analyses (Spearman Rho). 95 % confidence intervals (95 % CI) for the area under curve (AUC) in Receiver Operating Characteristics (ROC curves) were calculated nonparametricaly.
All statistical analyses were performed using GraphPad Prism Version 4.02.
The study population is described in table 1. The levels of sCD163 were measured on day 1 and day 3 of entrance. There was no difference in sCD163 levels between men and women (p=0.08 and p=0.95 for Day 1 and Day 3 respectively), and levels were not related to age (r=0.16, p=0.17 and r=−0.01, p=0.94 for day 1 and 3 respectively).
The median sCD163 levels were highly increased in patients with AFL on both day 1 (21.1 mg/l (range 3.6 – 74.9)) and day 3 (19.2 mg/l (2.6 – 67.2)) as compared to healthy controls (2.3 mg/l (0.65 – 5.6)) (p < 0.0001). Patients with chronic alcoholism and patients with stable alcoholic liver cirrhosis had intermediate levels (4.7 mg/l (2.9 – 11.0) and 9.8 mg/l (3.6 – 16.9) respectively) (Figure 1A). There was no statistical difference between CD163 levels day 1 and day 3 (p=0.68).
Grouping patients according to aetiology revealed that sCD163 in the acetaminophen group at day one was significantly lower than in patients with non-acetaminophen induced ALF (p=0.016). This difference, however, was levelled out at day 3. The highest concentrations were found at day one in patients with indeterminate causes (p=0.009 as compared to acetaminphen) (Figure 1B).
Significant correlations were found between sCD163 and ALT (r=−0.40, p=0.0004), AST (r=−0.33, p=0.005), bilirubin (r=0.41, p=0.0003), and creatinine (r=−0.23, p=0.046) at day 1, however these correlations were absent at day 3. No correlation was observed between sCD163 and INR. Patients with ascites at day 1 had higher CD163 levels (p=0.029), however no increase was seen in patients with peripheral oedema (p=0.53). Infected patients did not have increased levels of sCD163 (p=0.20). There was no correlation between the levels of sCD163 and the duration of icterus or the duration of symptoms. Patients with grade III or IV hepatic encephalopathy did not have higher levels of sCD163 than patients with lower grade encephalopathy.
The levels of sCD163 were compared between patients with spontaneous survival, patients who died (without transplantation), and patients who underwent liver transplantation. At day 1 the median levels in non-survivors were slightly increased as compared to survivors (23.5 mg/l (range 3.6 – 57.1) vs. 16.2 mg/l (5.6 – 74.9), p=0.087), and this difference was significantly expanded at day 3 (29.0 mg/l (7.2 – 54.0) vs. 14.6 mg/l (3.5 – 67.2), p=0.002) (Figure 2AB). When comparing the groups using the maximum sCD163 value for each patient who had both samples taken, the difference between non-survivors and survivors became even more pronounced (43.5 mg/l (7.2 – 57.1) vs. 21.1 mg/l (6.6 – 74.9), p= 0.0009) (Figure 2C). Patients that were transplanted had intermediate levels.
The ability of sCD163 to distinguish between non-survivors and survivors was described by generating Receiver Operating Characteristics (ROC curves) for sensitivity and specificity using different cut-off levels. At a cut-off level of 26 mg/l the sensitivity and specificity was 40.6 % and 75.0 % respectively at day 1 (Figure 3A) and 61.5 % and 81.3 % respectively at day 3 (Figure 3B). Using the maximum value of day 1 and 3, the sensitivity and specificity was 80.0 % and 72.7 % respectively (75 % and 81.8 % at a cut-off level of 27.5 mg/l) (Figure 3C).
In acute liver failure, the decision to perform a liver transplantation is a critical but difficult one. It is particularly challenging during the early phase of disease when chances for survival are best but the outcome most uncertain (8). At present the Kings College criteria (11) are most widely used, however, they lack sensitivity for accurate selection of patients at an early stage (8). New marker molecules that might be incorporated into more robust prognostic models therefore may have important clinical value.
In this report we have shown that the serum levels of soluble CD163, originating predominantly from anti-inflammatory macrophages, are increased 10-fold in acute liver failure compared to age and gender matched controls in agreement with previous findings in a smaller group of patients (7). Furthermore we have demonstrated that very high levels of sCD163 (> 26 mg/L) are related to poor disease outcome and may be valuable in selection of patients for transplantation.
The accuracy of measuring sCD163 alone for prediction of fatal outcome does not exceed what is obtained by traditional scores such as Kings College criteria (8), however, addition of sCD163 to Kings College criteria or other criteria such as MELD may improve sensitivity and identification of patients early in the disease process, which should be prospectively validated.
sCD163 was also elevated in patients with chronic alcoholism (2 times) and in patients with stable alcoholic liver cirrhosis (4–5 times), however still far from the very high levels found in ALF. None of the controls reached the levels associated with a poor prognosis in ALF.
CD163 is a marker-molecule in the plasma membrane of alternatively activated macrophages that is up-regulated by IL10 and glucocorticoid (12–14). The primary role for CD163 is the scavenging of haptoglobin-hemoglobin complexes (15), but it also appears to have anti-inflammatory activity (5). CD163 is almost exclusively restricted to monocytes and macrophages (16), and strong expression has been demonstrated in CD68-positive liver macrophages during acute hepatitis (6). Increased levels of IL10 in ALF have been reported in several reports (3,4), which may contribute to the very high levels of sCD163 in ALF.
Besides its presence in the membrane of macrophages, CD163 is found in the extra-cellular fluids (soluble CD163, sCD163), due to proteolytic cleavage of the membrane form by metalloproteinase activity (17). This shedding of sCD163 is dependent on activation of toll-like receptors by inflammatory mediators (18), that gives rise to increased levels of sCD163 in serum in diseases involving activation (e.g. hemophagocytosis, sepsis) and proliferation (e.g. leukaemia, Gaucher disease) of macrophages (19–22).
In vitro, macrophages can take up different phenotypes dependent on the cytokine environment (23,24), which is reflected in vivo by pro- and anti-inflammatory activation states balancing the immune responses. The high CD163 levels in ALF may represent an anti-inflammatory imbalance, especially in patients with a poor outcome of disease. This is supported by a recent report that describes a significant reduction in the expression of monocytic HLA-DR in patients with acute liver failure, and importantly, a correlation between monocytic deactivation and poor survival (3). The cells have impaired ability at presenting antigen and at producing TNF-alfa (3,23), and may represent a possible target for intervention.
A relation between high levels of sCD163 and mortality has also been described in patients with sepsis and hemophagocytosis (19,20). In sepsis, the levels of sCD163 are considerably lower than in ALF (e.g. 4.6 mg/l in pneumococcal baceteremia vs 21.1 mg/l in ALF (19)), but interestingly the relatively few sepsis-patients with very high sCD163 levels had increased mortality (19). Hemophagocytic syndrome is a clinicopathologic entity characterized by a generalized activation of macrophages with hemophagocytosis throughout the reticuloendothelial system, and clinical manifestations resulting from the overproduction of cytokines (25). The levels of sCD163 in hemophagocytosis are comparable to the very high levels found in ALF, whereas the levels seen in other situations of macrophage activation (infections, autoimmune disease, cancer) are considerably lower. This suggests that in ALF a large proportion of liver macrophages are activated and may contribute significantly to the pathogenesis of ALF.
Our study supports that sCD163 may be valuable as a disease marker. At day three of enrolment to the study, high levels of sCD163 were significantly associated with mortality. It is clear, however, that the time of sampling is crucial since the levels at day one was only marginally significant, and that the maximum level of sCD163 had the highest sensitivity and specificity for prediction of fatal outcome. Serial measurements of sCD163 should therefore be performed to evaluate if a continuous increase in sCD163 levels or a certain threshold may contain most information. It is interesting that sCD163 at day one correlated significantly to several biochemical liver-parameters, while this correlation was lost by day three, indicating an independent pathophysiological role for sCD163. Whereas the physiological roles for sCD163 in inflammatory diseases are poorly understood, the membrane bound sCD163 seems to strengthen the anti-inflammatory response by fueling heme oxygenase-1 and by upregulating its expression (5,26).
It is important to consider that the sequence of sCD163 concentrations may be different depending on the aetiology of ALF. This is emphasized by the low levels in the acetaminophen group and high levels in the group of patients with indeterminate causes at day 1. Whether differences in IL10 and other cytokines show the same pattern as the observed differences in sCD163 are presently being evaluated in the same cohort.
Since the shedding of soluble CD163 involves the loss of macrophage surface molecules, combined measurements of soluble CD163 and surface CD163 (by flow cytometry) would add more information on the regulation of CD163 in ALF, and the ratio between the two parameters possibly better predicts disease outcome (27).
The finding of successively increased levels in healthy, chronic alcoholics, stable alcoholic cirrhosis, and ALF may indicate a wider prognostic capability of sCD163, e.g. for the identification of alcoholic patients with incipient cirrhosis. This, however, was outside the scope of the present investigation.
In conclusion, the levels of soluble CD163 are 10 fold increased in patients with ALF, indicating that activated macrophages contribute significantly to the pathogenesis of ALF. Furthermore very high levels (> 26 mg/l) are correlated with mortality. Further study will be necessary to evaluate its use as a prognostic marker in predicting outcome in the setting of acute liver failure.
Funded by the Danish Medical Research Council (22-03-0355) and by NIH grant DK U-01 58369 for the Acute Liver Failure Study Group. Additional funding provided by the Tips Fund of Northwestern Medical Foundation and the Jeanne Roberts and Rollin and Mary Ella King Funds of the Southwestern Medical Foundation. Thanks to Kirsten Bank Petersen for excellent technical assistance.
The Acute Liver Failure Study Group 1998–2006 was comprised of the following investigators and coordinators who work tirelessly in support of this study: William M. Lee (PI), Julie Polson, Carla Pezzia, Ezmina Lalani, Linda S. Hynan, Joan S. Reisch, University of Texas Southwestern Medical Center, Dallas TX; Anne M. Larson, Hao Do, University of Washington, Seattle WA; Jeffrey S. Crippin, Laura Gerstle, Washington University School of Medicine, St. Louis MO; Timothy J. Davern, Katherine Partovi, University of California at San Francisco CA; Sukru Emre, Mt Sinai Medical Center, New York NY; Timothy M. McCashland, Tamara Bernard, University of Nebraska, Omaha NE; J. Eileen Hay, Cindy Groettum, Mayo Clinic, Rochester MN; Natalie Murray, Sonnya Coultrup, Baylor University Medical Center, Dallas TX; A. Obaid Shakil, Diane Morton, University of Pittsburgh Medical Center, Pittsburgh PA; Andres T. Blei, Jeanne Gottstein, Northwestern University Medical School, Chicago IL; Atif Zaman, Jonathan Schwartz, Ken Ingram, Oregon Health Sciences University, Portland OR; Steven Han, Val Peacock, University of California at Los Angeles, Los Angeles CA; Robert J. Fontana, Suzanne Welch, University of Michigan Medical Center, Ann Arbor MI; Brendan McGuire, Linda Avant, University of Alabama, Birmingham AL; Raymond Chung, Deborah Casson, Massachusetts General Hospital, Boston MA; Robert Brown Jr. and Michael Schilsky, Laren Senkbeil, Columbia-Presbyterian Medical Center/Cornell-New York Hospital, New York NY; M. Edwyn Harrison, Rebecca Rush, Mayo Clinic, Scottsdale, Scottsdale AZ; Adrian Reuben, Nancy Huntley, Medical University of South Carolina, Charleston SC; Santiago Munoz, Chandra Misra, Albert Einstein Medical Center, Philadelphia PA; Todd Stravitz, Jennifer Salvatori, Virginia Commonwealth University, Richmond VA; Lorenzo Rossaro, Colette Prosser, University of California, Davis Medical Center, Sacramento CA; Raj Satyanarayana, Wendy Taylor, Mayo Clinic, Jacksonville, Jacksonville FL; Raj Reddy, Mical Campbell, University of Pennsylvania, Philadelphia PA; Tarek Hassenein, Fatma Barakat, University of California, San Diego CA; Alistair Smith, Duke University, Durham NC.
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